Our long term goal is to understand the regulation of cell growth by cAMP-dependent protein kinase (PKA) by an in-depth investigation of the structure, function and expression of the regulatory (R) and catalytic (C) subunits of PKA. Two isoforms of PKA, PKA-I and PKA-II, share a common catalytic (C) subunit but contain distinct regulatory (R) subunits, RI and RII, respectively. It is known that expression of PKA-I and its regulatory subunit, RIa is increased in human cancer cell lines, in primary tumors, and in cells after transformation. Our studies revealed the following: (1) Use of antisense strategy: RIa antisense oligodeoxynucleotide or antisense RIa gene overexpression is used to demonstrate the sequence-specific inhibition of RIa gene expression and in vivo tumor growth inhibition. The loss of RIa by the antisense resulted in rapid increase in RIIb and PKA-IIb holoenzyme. Pulse-chase experiments demonstrated that RIIb protein increased its half-life 3-6 fold in antisense treated cells. Thus, RIIb in the holoenzyme complex is stabilized, exhibiting an increased half-life. Through this biochemical adaptation, in the antisense-treated cancer cells, the ratio of PKA-I to PKA-II changes to that similar to that of normal cells.; (2) Recombinant gene technology: Retroviral-vector mediated overexpression of wild type and point-mutated RIa, RIIa, RIIb and C subunits of PKA to demonstrate distinctive roles of the R subunit isoforms in cell growth, differentiation, and reverse transformation; and (3) Discovery of extracellular PKA (ECPKA):the free catalytic (C) subunit of PKA is excreted in conditioned medium of various cancer cells, and in serum of cancer patients; the ECPKA expression was upregulated 10-fold as compared with normal serum. Importantly, this study provided the means of modulating the ECPKA expression by changing expression of the intracellular PKA-I and PKA-II. Namely, (1) overexpression of RIa in expression vector, which upregulates intracellular PKA-I, can markedly upregulate ECPKA expression; (2) overexpression of RIIb, which downregulates PKA-I in the cell and reverts the transformed phenotype, downregulates ECPKA; and (3) A mutation in the Ca gene that prevents myristylation allows the intracellular PKA upregulation but blocks the ECPKA increase, suggesting that the N-terminal myristyl group of Ca is required for ECPKA expression. It was further shown that ECPKA upregulation is reduced in cancer cells maintaining hormone-dependency (a normal cell property), such as hormone-dependent breast cancer as compared to the hormone-independent breast cancer. These observations indicate that this phenomenon of ECPKA expression can provide innovative approach to cancer diagnosis, prognosis, and hormone-dependence detection (breast cancer).